Nonhuman model animal lacking the ability to control lymphocyte migration

ABSTRACT

The present invention provides a animal model useful in identifying a molecule controlling in a lymphocyte-specific manner migration and thus elucidating immune-related diseases and pathogenic conditions such as allergy, autoimmune diseases, GvH and graft rejections at a molecular level, or in developing a novel therapy. A nonhuman animal model such as a DOCK2 knockout mouse, in which the function to control lymphocyte migration has been deleted or suppressed, is generated by deleting DOCK2 gene on the chromosome. In this DOCK2 knockout mouse, the function of activating Rac to mediate actin cyteskeleton, the lymphocyte migration function in response to stimuli with chemokines such as SLC, SDF-1, BLC, the homing function to secondary lymphoid organs such as spleen, lymph nodes and Peyer&#39;s patches, and the function of emigrating mature thymic T cells into peripheral blood in response to stimulus with chemokine ELC are impaired, and as a result of this, immune responses are suppressed.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/973,432filed Oct. 9, 2007, abandoned, which is a divisional of U.S. applicationSer. No. 10/886,364 filed Jul. 7, 2004 and issued on Dec. 25, 2007 asU.S. Pat. No. 7,312,373, which is a continuation-in-part ofInternational Patent Application PCT/JP02/08372 filed Aug. 20, 2002,which claims priority from Japanese Patent Application JP 2002-707 filedJan. 7, 2002. Each of these applications, and each application andpatent mentioned in this document, and each document cited or referencedin each of the above applications and patents, including during theprosecution of each of the applications and patents (“application citeddocuments”) and any manufacturer's instructions or catalogues for anyproducts cited or mentioned in each of the applications and patents andin any of the application cited documents, are hereby incorporatedherein by reference.

Furthermore, all documents cited in this text, and all documents citedor referenced in documents cited in this text, and any manufacturer'sinstructions or catalogues for any products cited or mentioned in thistext, are hereby incorporated herein by reference.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. patent law; namely, that these terms are closed ended.

TECHNICAL FIELD

The present invention relates to a nonhuman animal model lacking thefunction to control lymphocyte migration whose function to controllymphocyte migration is lacked or suppressed by deleting DOCK2 gene, ahaematopoietic cell-specific CDM family protein, on the chromosome, amethod for screening promoters or suppressors of the function to controllymphocyte migration with the use of the model nonhuman animal, aprotein controlling lymphocyte migration which promotes reorganizationof cytoskeleton through activating Rac, or DNA encoding the proteincontrolling lymphocyte migration, or the like.

BACKGROUND ART

Immune-response is an indispensable defense mechanism against infectionfor a living body and immune cells are continuously patrolling within aliving body in order to rapidly cope with various sources of infection.This feature of the constituting cells to move around without cease isnot observed in other living complex systems and has been evolved as aunique feature to the immune system. It is known that, among immunecells, cells such as neutrophils and macrophages function at the earlydefense against infection whereas T and B lymphocytes induce theantigen-specific immune-response upon recognition of foreign substancesthrough their antigen receptors. Differentiation of such T and Blymphocytes takes place in primary lymphoid organs such as thymus andbone marrows. The differentiated lymphocytes then migrate to aparticular compartment in secondary lymphoid organs such as spleen,lymph nodes and Peyer's patches (lymphoid organ in the small intestine),where these lymphocytes induce specific immune-responses by recognizing,via the antigen receptors, antigens recruited from various tissues. Inthis process, it is considerably crucial for the establishment ofimmune-response that the lymphocytes migrate into a particular site insecondary lymphoid organs. Although lymphocyte migration has so far beenknown as being induced by proteins collectively referred to aschemokines of various kinds, the molecular mechanism controllinglymphocyte migration itself has been remained unknown.

Change in cellular polarization and reorganization of cytoskeleton isindispensable for cell migration (Cell 84, 359-369, 1996), both of whichhave been known to be regulated by low molecular-weight G proteins asRho, Rac and Cdc42 (Proc. Natl. Acad. Sci. USA 92, 5027-5031, 1995;Science 279, 509-514, 1998; J. Cell Biol. 141, 1147-1157, 1998; Science287, 1037-1040, 2000). Among these molecules, Rac yields driving forcefor cell mobility by forming actin-rich lamellipodial protrusion(Science 279, 509-514, 1998; Cell 103, 227-238, 2000). Meanwhile,molecules like CED5, DOCK180 and Myoblast city (MBC) that demonstratestructural homology have been identified for Caenorhabditis elegans,humans and Drosophila melanogaster, which molecules are called CDMfamily molecules with their acronyms and all of which are thought to beimplicated in reorganization of cytoskeleton by functioning in theupstream of Rac (Mol. Cell Biol. 16, 1770-1776, 1996; J. Cell Biol. 138,589-603, 1997; Nature 392, 501-504, 1998; Genes Dev. 12, 3331-3336,1998; Genes Dev. 12, 3337-3342, 1998; Nature Cell Biol. 2, 131-136,2000). Although genetic analysis using mutants has shown that the CED-5and Myoblast City are crucial for migration of particular types of cells(J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; NatureCell Biol. 2, 131-136, 2000), in what physiological way the CDM familyproteins function in mammals has been left unknown.

It is known than DOCK2 (KIAA0209; DNA Res. 3, 321-329) encodes other CDMfamily protein member specifically expressed in human haematopoieticcells and that the DOCK2 binds to Rac in 293T kidney cells foractivating Rac (Biochem. Biophys. Acta 1452, 179-187, 1999). On theother hand, the present inventors have found upon isolating a novel Hchgene belonging to CDM family from the mouse thymus cDNA library, thatthe Hch gene product is consisted of 1828 amino acids and SH3 domain isencoded at its N-terminus (Nature, Vol 412, 23 August, 826-831, 2001).Further, it was confirmed in northern blotting using mouse tissues thatHch expression is localized only in the thymus and spleen contrary tothe DOCK180 expression which is observed in various organs, and analysisusing cell lines provided confirmation that Hch expression is observedin all of T and B cells and macrophages except for two variant T celllines. Moreover, the present inventors have demonstrated that asignificant change in cell morphology as well as enhancement ofadhesiveness can be detected by introducing Hch into mutant T cell lineslacking Hch expression. Among the 1828 amino acids encoded by Hch, 1677amino acids were identical with those of human DOCK2, thus Hch wasthought to be mouse homologue of DOCK2, yet physiological function ofthe DOCK2 remained unrevealed.

Even though immune-response is an indispensable mechanism for a livingbody, diseases or pathogenic conditions developed as a result ofemergence of immune-response, for example autoimmune diseases, graftrejections, GvH, etc., are being focused as a problem which modernmedicine is expected to work out for the resolution. For elucidatingthese diseases or pathogenic conditions at a molecular level or fordeveloping a new therapy for these diseases or pathogenic conditions,molecules have been wanted to be identified that specifically controllymphocyte migration. The subject of the present invention is toidentify molecules controlling in a lymphocyte-specific mannermigration, and to provide useful animal models for elucidatingimmune-related diseases or pathogenic conditions such as allergy,autoimmune diseases, GvH or graft rejections at a molecular level, orfor developing a new therapy for these diseases or pathogenicconditions.

The present inventors isolated the CDM family DOCK2 (Hch) gene, which isspecifically expressed in the immune-system, from a mouse thymus cDNAlibrary, and generated the knockout mice in order to reveal the in vivofunction of the gene. DOCK2 knockout mice were born at the expectedmendilian ratio without apparent abnormality. However, the numbers of Tand B lymphocytes in secondary lymphoid organs such as spleen and lymphnodes were considerably reduced compared to those of wild-type mice.When lymphocytes labeled with fluorescence were intravenously injectedinto DOCK2 knockout mice for analyzing homing to lymph nodes, homingactivity of T and B lymphocytes of the knockout mice was reduced toaround 1/10 compared to that of wild-type mice. On the other hand, anefficient homing of lymphocytes to lymph nodes was observed in wild-typemice. These findings thus suggest that homing to secondary lymphoidorgans might be impaired in DOCK2 knockout mice owing to an intrinsicdefect in lymphocytes.

To address the involvement of DOCK2 molecules in lymphocyte mobility,migration activity in response to various chemokines were comparedbetween the knockout and wild-type mice. No difference was observedbetween the knockout and wild-type mice as to migration activity ofmacrophages to chemokines such as MCP-1 or SDF-1. However, contrary tothe fact that T and B lymphocytes from wild-type mice actively migratein response to chemokine stimuli such as with SLC, SDF-1 or BLC,migration of T and B lymphocytes from the knockout mice weresignificantly impaired. Upon stimulating lymphocytes of knockout micewith chemokines, Rac activation and actin polymerization were observedthat peaked at 15 sec. Such responses were disappeared in the knockoutmouse lymphocytes. Contrary, no difference was found between theknockout and wild-type mice as to PKB and ERK activations and calciumimmigration. These observations demonstrate that DOCK2 specificallycontrols lymphocyte migration by mediating reorganization ofcyteskeleton through activating Rac.

There was no marked abnormality in the differentiation of T and Blymphocytes in the primary lymphoid organs of DOCK2 knockout mice.Peripheral T lymphocytes from the knockout mice, however, weresignificantly decreased compared to that of the wild-type mice. Sincechemokine ELC are known to be involved in emigration of mature thymic Tcells from thymus, emigration of these T cells in response to ELC wasexamined by using thymus organ cultures. The results showed that theemigration efficiency of the knockout mice was reduced to about 1/20compared to that of the wild-type mice. These results suggest that theemigration defect in mature thymic T cells is responsible for decreasein the peripheral blood T lymphocytes of the knockout mice.

Chemokines such as SLC, ELC and BLC are called “immune-chemokines” andare known to play an essential role in architecture of secondarylymphoid organs. Marked atrophy of lymphoid follicles, straying oflymphocytes into red pulp, and disappearance of marginal-zone B cellswere observed in the immunohistological analysis for the spleen of DOCK2knockout mice. Atrophy of lymphoid follicles was similarly observed inother secondary lymphoid organs such as lymph nodes and Peyer's patches.Besides, marked aberration in distribution of mature thymic T cells inthe thymus of the knockout mice was found. It was thus suggested thatlymphocytes of DOCK2 knockout mice did not exercise migration activityin response to stimuli with various chemokines, leading to structuralabnormality in the immune system.

To study the influence of DOCK2 deficiency on immune-response, mice wereimmunized with Eα-derived peptide, which is known to bind to MHC classII I-A^(b), and the antigen-specific T-cell responses were analyzed. Asa result, proliferation response of T cells were observed in an antigenconcentration-dependent manner in wild-type mice, whereas such T cellresponse was not observed in DOCK2 knockout mice. DNP-KLH was alsoimmunized to mice to analyze the KLH-specific antibody production, andthe antibody production in DOCK2 knockout mice was observed to have beensignificantly impaired in DOCK2 knockout mice. These findings thussuggest that primary immune-response was suppressed in DOCK2 knockoutmice 7 days after the immunization.

Taking above findings together, it was demonstrated for the first timethat DOCK2 is an essential molecule controlling lymphocyte mobility bymediating reorganization of cyteskeleton through activating Rac, andthat DOCK2 deficiency largely affect architecture of the immune systemand immune-response. These findings are expected to serve for thedevelopment of new therapy for immune-related diseases such as allergy,autoimmune diseases and graft rejections by artificially controllinglymphocyte mobility with DOCK2 as a target. The present invention hasbeen completed based on these findings.

DISCLOSURE OF THE INVENTION

The present invention relates to a nonhuman animal model lacking thefunction to control lymphocyte migration wherein the function to controllymphocyte migration is lacked or suppressed by deleting DOCK2 gene onthe chromosome (“1”); the nonhuman animal model lacking the function tocontrol lymphocyte migration according to “1”, wherein the function tocontrol lymphocyte migration is a function to mediate cytoskeletalreorganization through activating Rac (“2”); the nonhuman animal modellacking the function to control lymphocyte migration according to “1”,wherein the function to control lymphocyte migration is a migrationfunction of lymphocytes in response to stimuli with chemokines such asSLC, SDF-1, BLC (“3”); the nonhuman animal model lacking the function tocontrol lymphocyte migration according to “1”, wherein the function tocontrol lymphocyte migration is a homing function to secondary lymphoidorgans such as spleen, lymph nodes and Peyer's patches (“4”); thenonhuman animal model lacking the function to control lymphocytemigration according to “1”, wherein the function to control lymphocytemigration is a function to emigrate mature thymic T cells intoperipheral blood in response to chemokine stimulus with ELC or afunction of intra-thymus migration of CD4⁺CD8⁺ immature thymocytes inresponse to chemokine stimulus with SDF-1 (“5”); the nonhuman animalmodel lacking the function to control lymphocyte migration according toany of “1” to “5”, wherein actin polymerization in response to chemokinestimulus is almost totally disappeared in lymphocytes (“6”); thenonhuman animal model lacking the function to control lymphocytemigration according to any of “1” to “6”, wherein marked atrophy oflymphoid follicles, straying of lymphocytes into red pulp, anddisappearance of marginal-zone B cells are observed (“7”); and thenonhuman animal model lacking the function to control lymphocytemigration according to any of “1” to “7”, wherein the nonhuman animal isa mouse (“8”).

The present invention also relates to a method for screening a promoteror suppressor of the function to control lymphocyte migration, wherein atest substance is administered to the nonhuman animal model lacking thefunction to control lymphocyte migration according to any of “1” to “8”,or a test substance is brought into contact with tissues, organs orcells from said nonhuman animal model and a wild-type animal tomeasure/assess the change in the function to control lymphocytemigration (“9”); the method for screening a promoter or suppressor ofthe function to control lymphocyte migration according to “9”, whereinthe change in the function to control lymphocyte migration is a changein the active GTP-bound Rac (“10”); the method for screening a promoteror suppressor of the function to control lymphocyte migration accordingto “9”, wherein the change in the function to control lymphocytemigration is a change in the migration activity of lymphocytes inresponse to stimuli with chemokines such as SLC, SDF-1, BLC (“11”); themethod for screening a promoter or suppressor of the function to controllymphocyte migration according to “9”, wherein the change in thefunction to control lymphocyte migration is a change in the homingactivity to secondary lymphoid organs such as spleen, lymph nodes,Peyer's patches and the like (“12”); the method for screening a promoteror suppressor of the function to control lymphocyte migration accordingto “9”, wherein the change in the function to control lymphocytemigration is a change in the number of mature T cells in peripheralblood in response to chemokine stimulus with ELC, or a change in theintra-thymic migration activity of CD4⁺CD8⁺ immature thymocytes inresponse to chemokine stimulus with SDF-1 (“13”); the method forscreening a promoter or suppressor of the function to control lymphocytemigration according to “9”, wherein the change in the function tocontrol lymphocyte migration is a change in the degree of actinpolymerization in lymphocytes in response to chemokine stimuli (“14”);the method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to “9”, wherein the change in thefunction to control lymphocyte migration is a change in the degrees ofatrophy of lymphoid follicles, straying of lymphocytes into red pulp,and disappearance of marginal-zone B cells (“15”); the method forscreening a promoter or suppressor of the function to control lymphocytemigration according to any of “9” to “15”, wherein the test substance isa molecule which binds to DOCK2 (“16”); the method for screening apromoter or suppressor of the function to control lymphocyte migrationaccording to any of “9” to “16”, wherein the degrees of change in thefunction to control lymphocyte migration of a nonhuman animal lackingDOCK2 gene on the chromosome and of a wild-type nonhuman animal arecompared and assessed (“17”); the method for screening a promoter orsuppressor of the function to control lymphocyte migration according toany of “9” to “17”, wherein the nonhuman animal is a mouse (“18”); apromoter or suppressor of the function to control lymphocyte migrationobtained by the method for screening according to any of “9” to “18”(“19”); the suppressor of the function to control lymphocyte migrationaccording to “19”, wherein said suppressor is an anti-DOCK2 antibody, aDOCK2-binding molecule or an antisense strand of DOCK2 gene (“20”); amethod for screening a therapeutic agent for immune-related diseasessuch as allergy, antoimmune diseases, GvH, graft rejections, wherein themethod for screening a promoter or suppressor of the function to controllymphocyte migration according to any of “9” to “18” is used (“21”) atherapeutic agent for immune-related diseases such as allergy,autoimmune diseases, GvH, graft rejections obtained by the screeningmethod according to “21” (“22”); and the therapeutic agent forimmune-related diseases according to “22”, which is an anti-DOCK2antibody, a DODK2-binding molecule or an antisense strand of DOCK2 gene(“23”).

The present invention further relates to a protein for controllinglymphocyte migration which mediates reorganization of cytoskeletonthrough activating Rac (“24”); the protein for controlling lymphocytemigration according to “24”, wherein said protein is DOCK2 and a DOCK2variant (“25”); the protein for controlling lymphocyte migrationaccording to “25”, wherein DOCK2 is an expression product of Hch gene(GenBank: accession No. AYO27438) (“26”); a method using the proteinaccording to any of “24” to “26” for controlling lymphocyte migration(“27”); DNA encoding a protein for controlling lymphocyte migrationwhich mediates reorganization of cytoskeleton through activating Rac(“28”); DNA encoding the protein for controlling lymphocyte migrationaccording to “28”, wherein said DNA is DOCK2 gene and a DOCK2 genevariant (“29”); DNA encoding the protein for controlling lymphocytemigration according to “29”, wherein DOCK2 gene is Hch gene (GenBank:accession No. AYO27438) (“30”); and a method using DNA according to anyof “28” to “30” for expressing the protein for controlling lymphocytemigration (“31”).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a series of photographs showing the results of reorganizationof actin cytoskeleton caused by DOCK2 in a T-cell line.

a: Total cell extracts were analyzed by immunoblotting using anti-DOCK2polyclonal antibody. A non-specific band (NS) is shown as a loadingcontrol.

b: Total cell extracts were analyzed by incubating with (bottom panel)or without (top panel) GST-fusion PAK1 RBD using anti-Rac monoclonalantibody.

c: Cells were stained with propidium iodide (PI) or phalloidin andanalyzed using a fluorescence microscope with Nomarski optics. Nomarski-and phalloidin-stained images were taken from the same cell.

d: After staining 25-7 cells with anti-HA antibody, DOCK2 (green area)and phalloidin (red area) were detected. The cell images were taken witha laser confocal microscope by scanning the cells from the top to thebottom (from 1 to 9) by a 2-μm interval. A scale bar in FIG. 1 dindicates 1 μm.

FIG. 2 is a series of graphs showing the results of examining the homingof DOCK2⁻/⁻ mouse-derived lymphocytes to secondary lymphoid organs.

a-c: Spleen and lymph node cells were stained to detect CD4, CD8, B220and Mac1. Figures (a), (b) and (c) show the number of total and eachsubset of cells in spleen, mesenteric lymph nodes and inguinal lymphnodes, respectively. Open bars in the figures denote cells from DOCK2⁺/⁻mice (n=4) and filled bars denote cells from DOCK2⁻/⁻ mice (n=7 for a,n=4 for b and c).d: Percentage of the fluorescence-labeled CD4⁺ T and B cells in theinguinal lymph nodes and spleen at 48 h of the transfer was analyzed.Open bars in the figures denote the results of transferring cells fromDOCK2⁺/⁻ mice to C57BL/6 mice (n=4), filled bars represent the resultsof transferring cells from DOCK2⁻/⁻ mice to C57BL/6 mice (n=5), andhatched bars represent the results of transferring cells from DOCK2⁺/⁻mice to DOCK2⁻/⁻ mice (n=5).e: Peripheral blood T or B cells were analyzed for the percentages (%)of the transferred cells 48 h after the transfer. Open bars in thefigure represent the results of transferring cells from DOCK2⁺/⁻ mice toC57BL/6 mice (n=7 for T cells, n=5 for B cells), and filled barsrepresent the results of transferring cells from DOCK2⁻/⁻ mice toC57BL/6 mice (n=6).

FIG. 3 is a photograph showing the analysis results of migrationactivity of the lymphocytes from DOCK2⁻/⁻ mice of the present invention.

a: A transwell chemotaxis assay was carried out by using spleen cellsfrom DOCK2⁺/⁻ mice (open bars) and DOCK2⁻/⁻ mice (filled bars). Theresults shown in the figures are expressed as the percentages to theinput cells.

b: Spleen cells were stained with anti-CXCR4 or anti-CCR7 antibody(Blood 96, 2074-2080, 2000) ([1]; red lines), or with monoclonalantibodies against Thy1.2 or B220 ([2]; green lines) as control, toanalyze receptor expression in Thy1.2⁺ T cells or B220⁺ B cells (graphsshow relative cell numbers).c: Splenic T and B cells (1.5×10⁷) treated with SDF-1 (500 ng/ml) forthe indicated times were analyzed for Rac activation using GST-fusionPAK1 RBD.d: Splenic T and B cells (5×10⁶) from DOCK2⁻/⁻ (▪) and DOCK2⁺/⁻ (●) micewere treated with SDF-1 (500 ng/ml) for the indicated times and analyzedfor the level of actin polymerization by flow cytometry. Results areexpressed as the average of mean channel fluorescence (MCF) in threeindependent experiments, with the baseline fluorescence assigned a valueof 100.e: Splenic B cells (5-8×10⁶) treated with SDF-1 (500 ng/ml) for theindicated times were analyzed for phosphorylation of PKB or ERK usingthe phospho-specific antibody against PKB (Ser⁴⁷³) or ERK1 and ERK2(Thr²⁰²/Tyr²⁰⁴), respectively.f: Fura-2-loaded splenic B cells were treated with SDF-1 at 250 ng/ml,and [Ca²⁺]_(i) was measured with a digital fluorescence microscopysystem.g: Signaling pathways from chemokine receptor to Rac activation wereschematized.

FIG. 4 is a series of photographs showing abnormal architecture of theimmune system in DOCK2⁻/⁻ mice of the present invention.

a: Sections for B cells (top panels) or T cells (bottom panels) werestained with anti-B220 antibody or anti-Thy1.2 monoclonal antibody,respectively.

b: Spleen sections were stained to detect B cells (green area), T cells(red area) and metallophilic macrophages (blue area) with anti-B220antibody, a mixture of anti-CD4 and anti-CD8 monoclonal antibodies, andMOMA1. The arrowhead indicates marginal-zone B cells.c: Spleen cells were stained to detect B220, CD23 and CD21/35, andB220⁺CD23⁻ B cells were analyzed for the CD21/35 expression (graph showsrelative cell numbers). Flow cytometric profiles (top panel) in thegated spleen cell population of DOCK2⁺/⁻ mice [[1] in the top panel (redline), and open bar in the bottom panel] and the spleen cell populationof DOCK2⁻/⁻ mice [[2] in the top panel (green line), and filled bar inthe bottom panel], and the percentage of CD21/35⁺ cells (bottom panel).d: Lymph node sections were stained to detect B cells (green area) and Tcells (red area) as in b.e: Peyer's patches were stained to detect B cells (green area) and Tcells (red area) as in b.f: Blood samples were counted and stained to detect CD4, CD8, B220 andMac1. The numbers of total and of each subset of peripheral bloodleukocytes are determined. Open bars in the figures show the results forDOCK2⁺/⁻ mice (n=4) and filled bars for DOCK2⁻/⁻ mice (n=4).g: Thymus organ cultures were used for transwell chemotaxis assays inthe presence of ELC. Figures on the left show expression of CD4 and CD8on the emigrated thymocytes and the figure on the right shows thenumbers of CD4⁺CD8⁻ and CD4⁻CD8⁺ mature thymocytes (SP) and CD4⁺CD8⁺thymocytes (DP) collected for from the thymus of DOCK2⁺/⁻ (open bars,n=3) and DOCK2⁻/⁻ mice (filled bars, n=3).h: Thymus sections were stained to detect CD4 (green area) and CD8 (redarea).i: Chemotactic response of CD4⁺CD8⁺ thymocytes to SDF-1 was measured andcompared between DOCK2⁺/⁻ (open bars) and DOCK2⁻/⁻ mice (filled bars)using a transwell chemotaxis assay. Results are expressed as percentages(%) of the input cells.

FIG. 5 shows suppression of immune-response in DOCK2⁻/⁻ mice of thepresent invention.

a: Mice were immunized with an antigenic peptide derived from Eαtogether with an adjuvant and CD4⁺ T cells were isolated from theregional lymph node 10 days after the immunization. The CD4⁺ T cellswere cultured in the presence of the peptide to analyze the T-cellproliferation. Vertical and horizontal bars show ³H-thymidine uptake andconcentration of Eα-derived antigenic peptide.b: DNP-KLH was immunized with an adjuvant and the blood was collected onday 7 of the immunization. Antigen-specific IgG in the serum wasqualified using an enzyme-antibody method. Vertical and horizontal barsshow OD450 levels and dilution system for the serum, respectively.

BEST MODE OF CARRYING OUT THE INVENTION

Any animal model whose function to control lymphocyte migration islacked or suppressed by deleting its DOCK2 gene on the chromosome may beused as a nonhuman animal model lacking the function to controllymphocyte migration. The function of DOCK2 gene can be deleted on itschromosome, for example, by inactivating the function by a gene mutationprocedure in which whole or part of a nonhuman animal endogenous geneencoding DOCK2 is disrupted, deleted or substituted and the like.Nonhuman animals for the present invention may include rodents such asmice, rats, etc. and also include rabbits, dogs, pigs, sheep, monkeys,etc. The nonhuman animals, of course, will not be limited to theseexamples, and mice are preferred among these examples because they areeasily handled and so on. As for the DOCK2 gene mentioned above, Hch(mouse DOCK2) gene (GenBank accession No. AYO27438; Nature, Vol 412, 23August, 826-831, 2001) and human DOCK2 gene (KIAA0209; DNA Res. 3,321-329) can be exemplified, yet the origin of DOCK2 gene is not limitedto mice, human and so on.

The function to control lymphocyte migration in the nonhuman animalmodel lacking the function to control lymphocyte migration according tothe present invention will not be limited to any particular function aslong as it is a function to control lymphocyte mobility affected by geneexpression of DOCK2. Yet the following functions are the examples; afunction to promote reorganization of cytoskeleton, particularly actinpolymerization in lymphocytes, through Rac activation for the formationof Rac-GTP complex, migration function of lymphocytes through stimuliwith chemokines such as SLC, SDF-1, BLC, etc., homing function tosecondary lymphoid organs such as spleen, lymph nodes and Peyer'spatches, emigration function of mature thymic T cells into peripheralblood in response to chemokine stimulus with ELC, migration function ofCD4⁺CD8⁺ immature thymocytes in response to chemokine stimulus withSDF-1, and so on. In the nonhuman animal models lacking the function tocontrol lymphocyte migration of the present invention in which thefunctions to control lymphocyte migration is deleted or suppressed,actin polymerization in lymphocytes in response to chemokine stimuli isalmost totally abolished, and these nonhuman animal models of thepresent invention are animal models, preferably DOCK2 knockout mice, inwhich marked atrophy of lymphoid follicles, straying of lymphocytes intored pulp, and disappearance of marginal-zone B cells are observed. Thefollowing is the description taking mice as an animal model.

A method for generating mice in which DOCK2 gene is deficient on itschromosome, i.e. DOCK2 knockout mice (DOCK2⁻/⁻ mice), will not belimited to a particular method and the mice can be generated by, forexample, a method previously described (Cell 76, 519-529, 1994) and thelike. For instance, DOCK2 gene is screened using gene fragments obtainedby PCR method or the like from the mouse genomic library. Whole or partof the screened DOCK2 gene is substituted by, e.g., a marker gene suchas neomycin-resistant gene using a recombinant DNA technology, then agene such as a diphtheria toxin A fragment (DT-A) gene or a herpessimplex virus thymidine kinase (HSV-tk) is introduced into the 5′terminal to obtain a targeting vector. The targeting vector thusobtained was linearized and introduced into ES cells by electroporationor the like to cause homologous recombination. ES cells resistant toantibiotics such as G418, gancyclovir (GANC) are selected from among thehomologous recombinants. The selected ES cells are preferably subjectedto southern blotting or the like to confirm whether they are therecombinants of the target.

The recombinant ES cells above are microinjected into mouse blastocystsand the blastocysts are transplanted to recipient mice to generatechimeric mice. Intercrossing of the chimeric and wild-type mice givesheterozygous mice, and further intercrossing of these heterozygous micegives DOCK2 knockout mice. Whether DOCK2 gene is actually deficient onthe chromosome in such DOCK2 knockout mice can be confirmed by methodslike southern blotting wherein isolated DNA from tails of the mice thusobtained is analyzed, immunoblotting wherein proteins extracted fromlymphocytes or the like of the mice are analyzed, and so on.

Nonhuman animal models lacking the function to control lymphocytemigration of the present invention are highly useful as animal modelsfor elucidating immune-related diseases and pathogenic conditions suchas allergy, autoimmune diseases, GvH and graft rejections, or as animalmodels for developing a new therapy for these diseases and pathogenicconditions. Comparison between mice lacking the function to controllymphocyte migration and wild-type mice with the use of the nonhumananimal models lacking the function to control lymphocyte migrationenables to screen promoters or suppressors of the function to controllymphocyte migration with DOCK2 as a target and to screen a therapeuticagent for immune-related diseases such as allergy, autoimmune diseases,GvH and graft rejections.

Method for screening promoters or suppressors of the function to controllymphocyte migration of the present invention include a method wherein atest substance is administered to the aforementioned nonhuman animalmodel lacking the function to control lymphocyte migration of thepresent invention, and a method wherein a test substance and tissues,organs or cells of a nonhuman animal model lacking the function tocontrol lymphocyte migration are brought into contact. As a method tobring a test substance and tissues, organs or cells of a nonhuman animalmodel lacking the function to control lymphocyte migration, e.g. theDOCK2 knockout mouse aforementioned, and a wild-type animal intocontact, there is a method to bring a test substance and tissues, organsor cells of these animals into contact, and to determine/assess changein the function to control lymphocyte migration such as changes inactive Rac bound to GTP, in cellular polarity such as uneven nucleardistribution, in actin polymerization, in migration activity oflymphocytes in response to stimuli with chemokines such as SLC, SDF-1and BLC. Examples of methods for administrating a test substance to anonhuman animal model lacking the function to control lymphocytemigration, e.g. the aforementioned DOCK2 knockout mouse, and to awild-type animal include a method for measuring/assessing changes in thefunction controlling lymphocyte migration in the tissue, organ or cellsof the mice, for example, change in active GTP-bound Rac in cells suchas lymphocytes, change in cellular polarization such as uneven nucleardistribution, change in actin polymerization, change in migrationactivity of lymphocytes in response to stimuli with chemokines such asSLC, SDF-1 and BLC in the mice, change in homing activity into secondarylymphoid organs such as spleen, lymph nodes and Peyer's patches, changein the number of mature T cells in peripheral blood in response tostimulus with chemokine ELC, change in migration activity of CD4⁺CD8⁺immature tymocytes in peripheral blood in response to stimulus withchemokine SDF-1, or the degrees of change in atrophy of lymphoidfollicles, straying of lymphocytes into red pulp, and disappearance ofmarginal-zone B cells. The methods, however, will not be limited only tothese examples.

In performing screening described above, promoters or suppressors of thefunction to control lymphocyte migration, especially suppressors, can beefficiently screened by using molecules binding to DOCK2 in vitro or thelike, preferably molecules specifically binding to DOCK2, as a testsubstance. Methods for searching molecules binding to DOCK2 in vitro orthe like preferably include those conventionally known search methodsfor interacting proteins such as two-hybrid system using yeast, farwestern method using E. Coli expression system, immunoprecipitationmethod, and a method using affinity chromatography. Further, inperforming the above-described screenings, it is preferable tocompare/assess the degree of change in the function to controllymphocyte migration of nonhuman animals lacking DOCK2 gene on thechromosome and the degree of change in that of wild-type nonhumananimals, preferably in that of their littermate nonhuman animals.

With the use of the above-described methods for screening promoters orsuppressors of the function to control lymphocyte migration, screeningfor therapeutic agents for treating immune-related diseases such asallergy, autoimmune diseases, GvH and graft rejections can be madepossible. For instance, suppressors of the function to controllymphocyte migration including anti-DOCK2 antibody, DOCK2-bindingmolecule, an antisense strand of DOCK2 gene obtained by the method forscreening promoters or suppressors of the function to control lymphocytemigration are expected to artificially suppress lymphocyte trafficking,so that these suppressors are highly promising for therapeutic agentsfor treating immune-related diseases such as allergy, autoimmunediseases, GvH and graft rejections. When therapeutic agents are used asmedical products, various compounding components for formulation can beadded such as common carriers, binders, stabilizers, excipients,dilutions, pH-buffers, disintegrants, solubilizers, solubilizingadjuvants, isotonic agents, that are pharmaceutically acceptable. Thetherapeutic agents can be administered in an usually used formulation,for example by oral administration in forms of powders, granules,capsules, syrups, suspensions or the like, or by parenteraladministeration in the form of injections formulated to solutions,emulsions, suspensions, etc.

The present invention targets proteins controlling lymphocyte migrationthat promote reorganization of cytoskeleton through activating Rac, orDNAs encoding such proteins controlling lymphocyte migration. Thepresent invention also targets the use of the proteins controllinglymphocyte migration that promote reorganization of cytoskeleton throughactivating Rac for the purpose of controlling lymphocyte migration, anda method using DNAs encoding the proteins controlling lymphocytemigration for the purpose of expressing the proteins controllinglymphocyte migration. Specific examples of such proteins or DNAs areDOCK2 such as Hch, or DOCK2 gene such as Hch gene (GenBank: accessionNo. AYO27438). As described in the above, the function of the proteinscontrolling lymphocyte migration that promote reorganization ofcytoskeleton through Rac activation, or the function of DNAs encodingsuch proteins controlling lymphocyte migration have never been revealedbefore having elucidated by the present invention.

The present invention will be explained more specifically with referenceto the following examples, the technical scope of the invention,however, will not be limited to these exemplifications.

Example 1 Reorganization of Actin Cytoskeleton Mediated by DOCK2 in theT-Cell Line

DOCK2 gene (KIAA0209; DNA Res. 3, 321-329) is known to encode CDM familyproteins specifically expressed in human haematopoietic cells. AlthoughDOCK2 has been shown to bind to and activate Rac in 293T kidney cells(Biochem. Biophys. Acta 1452, 179-187, 1999), its physiological functionremained unknown.

In an attempt to search the genes that are expressed in the mousethymus, the present inventors isolated complementary DNA, designated asHch gene (GenBank accession number: AYO27438), which predictably encodes1828 amino acids including SH (Src-homology)-3 domain at the aminoterminus and found that Hch was homologous to CED-5, MBC and DOCK180;furthermore, 1677 of the 1828 amino acids of Hch were identical to humanDOCK2. Whereas DOCK180 was expressed in various tissues, the expressionof Hch was restricted to thymus, spleen and lymph nodes, suggesting thatHch is specifically expressed in haematopoietic cells. The Hchexpression was detected in all T-, B- and monocyte cell lines tested,with the exception of two T-cell lines, BW5147α⁻β⁻ and its derivativeBEα16-3 (referred to as “16-3” hereinafter). From these results, thepresent inventors concluded that Hch gene is a mouse homologue of humanDOCK2 gene.

Whether the expression of DOCK2 gene affects the actin cytoskeleton wasfirst examined by introducing mouse DOCK2 gene into the T-cell line 16-3which lacks DOCK2 gene. As a result of western blot analysis, two celllines stably expressing transgenes, 17-11 and 25-7, showed slightlyhigher (17-11) or lower (25-7) expression compared with a wild-type cellline N3-5 (FIG. 1 a). The active GTP-bound Rac, which interacts withPAK1 (Nature 367, 40-46, 1994), was detected with 17-11 and N3-5, butnot with 16-3 (FIG. 1 b). In several aspects, the present inventorsfound that cell lines 17-11 and 25-7 were morphologically different from16-3, but were similar to N3-5. Although 16-3 had a round cell shape,17-11, 25-7 and N3-5 showed a relatively flat and polarized cell shapewith a biased localization of the nucleus (FIG. 1 c). When cells werestained with phalloidin, F-actin assembly was observed in 17-11, 25-7and N3-5, but not in 16-3 (FIG. 1 c). The confocal laser microscopicanalysis revealed that the introduced DOCK2 did not co-localize with,but was located adjacent to, this F-actin assembly at the root of thelamellipodial protrusion (FIG. 1 d). Thus it was revealed that, likeother CDM family proteins, DOCK2 mediates reorganization of actincytoskeleton in T cells through Rac activation.

Example 2 Generation of DOCK2⁻/⁻ Mice

Genomic clones containing the mouse DOCK2 gene were isolated from a129/Sv genomic library. The targeting vector was constructed byreplacing a 3.1 kb fragment from the KpnI site located within exon 3 tothe BamHI site between exon 5 and 6 with a neomycin resistance cassette(neo). This vector was linearized and subjected to electroporation, thenintroduced into ES cells to generate homologous recombinants. ES cloneswere isolated from these homologous recombinants, theneomycin-resistance ES clones were then screened by using G418. Thehomologous recombinants were confirmed by southern blotting. GenomicDNAs were isolated from these homologous recombinants and were digestedwith Sph I and Eco RI, then the DNAs were confirmed to have beenreplaced by targeting alleles containing neo cassettes. After theconfirmation, the targeted ES clones were microinjected into C57BL/6blastocysts and the male chimeric mice obtained were crossed withC57BL/6 female mice. The heterozygous mice (DOCK2⁺/⁻) with mutantalleles were intercrossed to obtain DOCK2⁻/⁻ mice. Southern blotanalysis confirmed that wild-type DOCK2 alleles were specificallydisrupted in mutant mice (DOCK2⁺/⁻ mice). RT-PCR (polymerase chainreaction with reverse transcription) revealed that insertion of the neocassette results in the appearance of a stop codon at the N-terminusportion of DOCK2, leaving only 42 amino-acid residues. DOCK2 expressionwas not detected in the spleen and thymus of mutant mice in a westernblot analysis. In the following experiments, the DOCK2⁺/⁻ and DOCK2⁻/⁻littermate mice were used.

Example 3 Defect in Homing of DOCK2⁻/⁻ Mouse-Derived Lymphocytes toSecondary Lymphoid Organs

To investigate the physiological function of DOCK2, the presentinventors generated DOCK2-deficient mice by homologous recombination inembryonic stem (ES) cells. DOCK2⁻/⁻ mice were born at the expectedmendelian ratio without apparent abnormality. However, the total numberof spleen cells in DOCK2⁻/⁻ mice was reduced to about 50% of that inDOCK2⁺/⁻ mice (FIG. 2 a). Although the proportion of CD4⁺ and CD8⁺ Tcells, and also B cells, were not much changed in DOCK2⁻/⁻ mice fromthat of DOCK2⁺/⁻ mice in the spleen cells, the proportion of Mac1⁺monocytes was significantly increased in DOCK2⁻/⁻ mice. A similartendency was observed with mesenteric and inguinal lymph node cells,except for the proportion of CD4⁺ T cells (FIG. 2 b, c): Whereas CD4⁺ Tcells occupied approximately 40% of lymph node cells in DOCK2⁺/⁻ mice,this proportion decreased to less than 25% in DOCK2⁻/⁻ mice.Collectively, the total numbers of T- and B-cells, but not monocytes,were markedly reduced in the spleen and lymph nodes of DOCK2⁻/⁻ mice.

These findings raised the possibility that lymphocyte homing tosecondary lymphoid organs might be impaired in DOCK2⁻/⁻ mice. To addressthis issue, the present inventors compared and investigated chemotaxisof fluorescence-labeled CD4⁺ T- or B-cells to the peripheral lymph nodesor spleen. When cells prepared from DOCK2⁺/⁻ mice were injectedintravenously into wild-type mice (C57BL/6), 0.34 and 0.05% of theinjected CD4⁺ T- or B-cells, on average, migrated to the inguinal lymphnodes, respectively (FIG. 2 d, left). However, when cells prepared fromDOCK2⁻/⁻ mice were injected to wild-type mice, the percentages of themigrated CD4⁺ T- or B-cells were reduced to about 10% of the levelobserved upon injecting cells from DOCK2⁺/⁻ mice. A similar differencewas found on analysis of migration to the wild-type spleen (FIG. 2 d,right). On the other hand, CD4⁺ T- and B-cells prepared from DOCK2⁺/⁻mice efficiently migrated to the inguinal lymph nodes and spleen ofDOCK2⁻/⁻ mice (FIG. 2 d). These results suggested that lymphocyte homingis impaired in DOCK2⁻/⁻ mice owing to an intrinsic defect inlymphocytes. Although no significant differences were found in theexpression frequency of transferred T cells in the peripheral blood ofwild-type mice, the percentage of the transferred mouse DOCK2⁻/⁻ B cellsshowed a 2.7-fold increase in level compared with the mouse DOCK2⁺/⁻ Bcells (FIG. 2 e).

Example 4 Defect in Migration Activity in DOCK2⁻/⁻ Mice-DerivedLymphocytes

What was analyzed next was chemotaxis of splenic T- and B cells inresponse to stimuli with several chemokines, such as secondary lymphoidorgan chemokine (SLC), B-lymphocyte chemoattractant (BLC),stroma-derived factor (SDF)-1 and monocyte chemotactic protein (MCP)-1.Whereas T- and B-cells from DOCK2⁺/⁻ mice efficiently migrated inresponse to SLC, SDF-1 and BLC, those from DOCK2⁻/⁻ mice did not showany response to stimuli with these chemokines (FIG. 3 a, left andmiddle). In contrast, chemotactic response was observed in the DOCK2⁻/⁻mouse monocytes in response to stimuli with MCP-1 and SDF-1, and asimilar chemotactic response was also observed in monocytes fromDOCK2⁺/⁻ mice (FIG. 3 a, right). Since some reports demonstrate that thechemotactic activities of SLC and SDF-1 are induced through interactionwith their receptors, CCR7 or CXCR4, respectively (Nature 382, 829-833,1996; Nature 382, 833-835, 1996; J. Biol. Chem. 273, 7118-7122, 1998),the expressions of CXCR4 or CCR7 on the surface of splenic B cells andon splenic T cells were analyzed. The result showed that splenic B cellsfrom DOCK2⁻/⁻ and DOCK2⁺/⁻ mice comparably expressed CXCR4 on theirsurface, and that the expressions of CXCR4 and CCR7 on splenic T cellswere even higher in DOCK2⁻/⁻ than in DOCK2⁺/⁻ mice (FIG. 3 b). Thus itis clear that the defect in chemotactic response of DOCK2⁻/⁻ lymphocytesdoes not result from extremely low, or absence of, receptor expression.Taken together, these results indicate that although DOCK2 is notinvolved in monocyte migration, DOCK2 molecules are indispensable forlymphocyte migration by functioning in the downstream of chemokinereceptors.

Chemokine receptors are coupled with heterodimeric G_(i) proteins thatactivate a variety of signaling pathways. Phosphatidylinositol-3-OHkinases (PI(3)K) in the downstream of the chemokine receptors areessential signaling molecules involved in activation of Rac, proteinkinase B (PKB) and extracellular signal-regulated kinases (ERKs)(Immunol. Rev. 177, 217-235, 2000, Nature Immunol. 2, 129-134, 2001).When T cells and B cells from DOCK2⁺/⁻ mice were stimulated with SDF-1,the activated Rac was detected. Such activation, however, was totallyabolished in DOCK2⁻/⁻ lymphocytes (FIG. 3 c). Consistent with thisresult, SDF-1-induced actin polymerization was observed in DOCK2⁺/⁻lymphocytes, but was scarcely detected in DOCK2⁻/⁻ lymphocytes (FIG. 3d). In contrast, SDF-1-induced phosphorylations of PKB, ERK1 and ERK2were comparable between both DOCK2⁺/⁻ and DOCK2⁻/⁻ B cells (FIG. 3 e).It is known that Chemokines can also activate phospholipase C leading toan increase in intracellular Ca²⁺ concentration [Ca²⁺]_(i) (NatureImmunol. 2, 129-134, 2001). As a result of examining inflow of Ca²⁺induced by SDF-1, such inflow was revealed to occur to a similar extentin both DOCK2⁺/⁻ and DOCK2⁻/⁻ B cells (FIG. 3 f). These results indicatethat in chemokine receptor-mediated signaling pathways DOCK2 ispredominantly involved in Rac activation (FIG. 3 g).

Example 5 Abnormal Architecture of the Immune System in DOCK2⁻/⁻ Mice

The immuno-chemokines such as SLC and BLC are known to be critical fortrafficking of lymphocytes into subcompartments of secondary lymphoidorgans (Cell 87, 1037-1047, 1996, Blood 91, 2886-2895, 1998, Cell 99,23-33, 1999, J. Exp. Med. 189, 451-460, 1999). As lymphocytes fromDOCK2⁻/⁻ mice lacked chemotactic response to these chemokines in vitro,spleen, lymph nodes and Peyer's patches were also examined. In thespleen of DOCK2⁺/⁻ mice, lymphoid follicles that consisted of T-cellzone and the surrounding B-cell area were clearly defined (FIG. 4 a,left). However, such lymphoid follicles were quite atrophic in theDOCK2⁺/⁻ mouse spleens, and scattered distribution of lymphocytes (T andB cells) in red pulp was observed in this line (FIG. 4 a, right).Immunofluorescence analysis of splenic tissue sections revealed thatmarginal-zone B cells, normally distributed around metallophilicmacrophages, were markedly reduced in DOCK2⁻/⁻ mice (FIG. 4 b). Themarginal-zone B cells can be characterized asCD21/CD35^(high)CD23^(neg-low) B cells (Eur. J. Immunol. 27, 2366-2374,1997). Although around 25-30% of B220⁺CD23⁻ splenic B cells expressedCD21/CD35 in DOCK2⁺/⁻ mice, such population of B cells was scarcelyfound in DOCK2⁻/⁻ mice, confirming the defect of marginal-zone B cellsin DOCK2⁻/⁻ line (FIG. 4 c). The atrophy of lymphoid follicles andaberrant T-cell distribution were also observed in lymph nodes ofDOCK2⁻/⁻ mice (FIG. 4 d). In addition, it was also found that Peyer'spatches were poorly developed in DOCK2⁻/⁻ mice in terms of seize andcell density (FIG. 4 e).

SDF-1 serves as a chemoattractant for immature lymphocytes. Thechemotactic responses of DOCK2⁻/⁻ pro-B and pre-B cells to SDF-1 (250ng/ml) were significantly reduced compared with those of DOCK2⁺/⁻ mice(3.4% versus 18.5% in pro-B cells; 0.5% versus 5.6% in pre-B cells,respectively). However, the amounts of pro-B cells, pre-B cells,immature B cells and myeloid cells in the bone marrow were unchangedbetween in DOCK2⁺/⁻ and DOCK2⁻/⁻ mice. This indicates that DOCK2deficiency affects neither B lymphopoiesis nor myelopoiesis. Althoughthe total number of thymocytes and the proportion of CD4⁻CD8⁻ thymocytesin DOCK2⁻/⁻ mice decreased and increased, respectively, compared withthose in DOCK2⁺/⁻ mice, the proportion of mature thymocytes in theselines were comparable. Nonetheless, the total number of CD4⁺ and CD8⁺ Tcells in peripheal blood decreased extremely in DOCK2⁻/⁻ mice (FIG. 4f). Thymocyte migrations in DOCK2⁺/⁻ and DOCK2⁻/⁻ mice in response toEBI1-ligand chemokine (ELC), which is known to function as achemoattractant for mature thymocytes, were compared (Blood 91,4434-4443, 1998). Although CD4⁺CD8⁻ and CD4⁻ CD8⁺ mature thymocytes fromthe DOCK2⁺/⁻ thymus efficiently emigrated, mature thymocytes werescarcely detected in the case of DOCK2⁻/⁻ mice (FIG. 4 g, left). Theefficacy of mature thymocyte emigration from the thymus of DOCK2⁻/⁻ micewas reduced to less than 5% of the wild-type level (FIG. 4 g, right). Itis thus suggested that an emigration defect of mature thymocytes to theperipheral bloodstream is responsible for T lymphocytopenia observed inDOCK2⁻/⁻ mice. It was confirmed that in the thymus of DOCK2⁻/⁻ mice,CD4⁺CD8⁻ and CD4⁻CD8⁺ mature thymocytes were distributed irregularlythroughout the thymus as small patches (FIG. 4 h). Although the precisemechanism for this currently remains unknown, thymocyte traffickingwithin the thymus may also be impaired in DOCK2⁻/⁻ mice. This could alsobe evidenced by the fact that the chemotactic response of CD4⁺CD8⁺immature thymocytes to SDF-1 was severely impaired in DOCK2⁻/⁻ mice(FIG. 4 i).

Example 6 Defect in Immune-Response of DOCK2⁻/⁻ Mice

To assess the influence of DOCK2 defect on immune-response, DOCK2⁻/⁻ andDOCK2⁺/⁻ mice were immunized at their tail undersides with 50 g ofEα-derived peptide, which is known to bind to MHC class II I-A^(b),together with an adjuvant. Ten days after the immunization, CD4⁺ T cellswere isolated from the regional lymph node and cultured in the presenceof Eα-derived peptide in vitro to analyze the antigen-specific T cellresponse. T cells were observed to proliferate in a peptideconcentration-dependent manner in wild-type mice, whereas in DOCK2⁻/⁻mice no such response of T cells was observed. Further, DNP-KLH wassimilarly immunized to analyze production of KLH-specific antibodies, asa result, the antibody production in DOCK2⁻/⁻ mice was significantlyimpaired on day 7 after the immunization. That is to say, it wasrevealed that primary immune-response was impaired in DOCK2⁻/⁻ mice.

These observation results demonstrate that haematopoietic cell-specificCDM family protein DOCK2 functions as a main molecule that activates Racand mediates cytoskeletal reorganization in lymphocyte migration.Several abnormalities that were observed in DOCK2⁻/⁻ mice were similarto those of mice that lacked CCR7, SLC or CXCR5 (the receptor cells forBLC) (Cell 87, 1037-1047, 1996, Blood 91, 2886-2895, 1998, Cell 99,23-33, 1999, J. Exp. Med. 189, 451-460, 1999). However, DOCK2⁻/⁻ miceexhibited abnormalities in a broader range in that they showedphenotypes that are not observed in the above-mentioned deficient micesuch as T lymphocytopenia, loss of marginal-zone B cells, abnormalthymus architecture and reduced lymphocyte homing to the spleen. Some ofthese features probably reflect unresponsiveness of lymphocytes fromDOCK2⁻/⁻ mice to other unknown chemokines or chemokines having undefinedfunction. Furthermore, because of its nature to regulate the actincytoskeleton, DOCK2 may not merely be involved in chemokine-mediatedlymphocyte migration but also in other higher functions of lymphocytes.

Method 1 (Preparation of Cells that Stably Express Transgenes)

A full-length DOCK2 gene, or a full-length DOCK2 gene fused with aninfluenza hemagglutinin (HA) peptide tag was inserted into aPBJ1-expressing vector and transferred to 16-3T cell lines byelectroporation.

Method 2 (Immunoblot Analysis)

Anti-mouse DOCK2 polyclonal antibody was developed by immunizing rabbitswith carboxy-terminal peptide of DOCK2 to which a keyhole limpethaemocyanin is conjugated, and this was used to detect DOCK2 expression.To assess Rac activation, cell extracts were incubated in the presenceof the glutathione S-transferase (GST)-fusion and Rac-binding domain(RBD) of PAK1, and subjected to immunoblot analysis using anti-Racmonoclonal antibody (Upstate Biotechnology). Activity of PKB or ERK wasassessed with phospho-specific antibodies against PKB (Ser⁴⁷³) or ERK1and ERK2, (Thr²⁰²/Tyr²⁰⁴), respectively (Cell Signalling).

Method 3 (Immunofluorescent Microscopy)

Cells were fixed with 4% formaldehyde in phosphate-buffered saline(PBS), permeabilized with 0.1% saponin and stained with propidium iodide(CALBIOCHEM), Alexa 568-labeled phalloidin (Molecular Probes) and/oranti-HA antibody (Santa Cruz) followed by Alexa 488-labeled anti-rabbitimmunoglobulin (Ig)-γ. The staining profiles were visualized with eithera fluorescence microscope equipped with Nomarski optics or a confocallaser-scanning microscope equipped with an argon/krypton laser capableof dual excitation.

Method 4 (Blood Counts and Flow Cytometric Analysis)

Blood was obtained by retro-orbital venous plexus puncture and subjectedto an analysis on an automatic cell counter. Cell counts were determinedfor bone marrow cells, thymocytes, spleen cells and lymph node cellsusing Neubauer chambers. Flow cytometry was carried out by staining thecells with the relevant monoclonal antibodies (PharMingen) and analyzingthem on a FACScan (Becton-Dickinson). To assess actin polymerization,purified T and B cells were fixed with 4% paraformaldehyde in PBS,treated with 0.1% saponin, and stained with Alexa 488-labeled phalloidinfor flow cytometry.

Method 5 (Tissue Staining)

Cryostat sections were fixed in acetone and 1% paraformaldehyde, andstained with Alexa 488- or 594-labeled monoclonal antibodies. Stainingwith anti-metallophilic macrophage monoclonal antibody (MOMA1) wasfollowed by reaction with biotinylated anti-rat IgG antibody and reactedand stained with streptavidin-conjugated Cy5.5. In some experiments,tissue sections were stained with biotinylated monoclonal antibodies andvisualized with a Vectastain ABC-PO kit (Vector Laboratories).

Method 6 (Lymphocyte Homing Assay)

Purified CD4⁺ T or B cells (4×10⁷ ml) were labeled with 3 μM BCECF-AMsolution (Dojindo), washed with PBS and intravenously injected into mice(8×10⁶ to 1×10⁷ per mouse). After 48 h, cells were prepared from thespleen and inguinal lymph nodes, and counted. Before and after the celltransfer, cells were stained with anti-CD4 or anti-B220 monoclonalantibody, and the percentage of migrated cells (%) was calculated. Thepercentages of transferred T and B cells in peripheral blood werecalculated by staining the cells with anti-Thy1.2 or anti-B220monoclonal antibody 48 after the transfer.

Method 7 (Chemotaxis Assay)

Thymus organ, spleen cells (1.5×10⁶), bone marrow cells (1.5×10⁶) andthymocytes (1.5×10⁶) in 100 μl RPMI medium were loaded into transwells(Coaster, 5-μm pore size), which were placed onto 24-well platescontaining 450 μl RPMI medium supplemented with chemokines(Genzyme/Techne) at various concentrations. After 3 h of incubation at37° C., cells migrated to the lower chamber were collected and stainedwith the relevant antibodies. Cells were counted with a FACScan.

Method 8 (Measurement of Intracellular Ca²⁺ Concentration)

[Ca²⁺]_(i) was measured using the Attofluor (fluorescent dye) digitalfluorescence microscopy system (Carl Zeiss). Cells loaded with fura-2/AM(Dojindo) were put in a chamber of 0.5-ml volume and mounted on aninverted microscope. The measurement of intracellular Ca2⁺ concentrationwas performed while the bath was continuously perfused with a modifiedKrebs solution (pH 7.3). The fura-2 fluorescence images were recordedinto a re-writable optical disc recorder at a rate of roughly 1 Hz andconverted to Ca²⁺ concentration.

Method 9 (Analysis of Immune-Response)

Mice were immunized with an antigenic peptide together with an adjuvantat the underside of tails, and CD4⁺ T cells were isolated from theregional lymph node and cultured in the presence of the antigenicpeptide. ³H-thymidine uptake was then measured to determine theproliferation of T cells. Blood was collected after the immunization andthe antigen-specific IgG antibodies were quantified with anenzyme-antibody method.

The invention will now be further described by the following numberedparagraphs:

1. A nonhuman animal model lacking the function to control lymphocytemigration wherein the function to control lymphocyte migration is lackedor suppressed by deleting DOCK2 gene on the chromosome.

2. The nonhuman animal model lacking the function to control lymphocytemigration according to paragraph 1, wherein the function to controllymphocyte migration is a function to mediate cytoskeletalreorganization through activating Rac.

3. The nonhuman animal model lacking the function to control lymphocytemigration according to paragraph 1, wherein the function to controllymphocyte migration is a migration function of lymphocytes in responseto stimuli with chemokines such as SLC, SDF-1, BLC.4. The nonhuman animal model lacking the function to control lymphocytemigration according to paragraph 1, wherein the function to controllymphocyte migration is a homing function to secondary lymphoid organssuch as spleen, lymph nodes and Peyer's patches.5. The nonhuman animal model lacking the function to control lymphocytemigration according to paragraph 1, wherein the function to controllymphocyte migration is a function to emigrate mature thymic T cellsinto peripheral blood in response to chemokine stimulus with ELC or afunction of intra-thymus migration of CD4⁺CD8⁺ immature thymocytes inresponse to chemokine stimulus with SDF-1.6. The nonhuman animal model lacking the function to control lymphocytemigration according to any of paragraphs 1 to 5, wherein actinpolymerization in response to chemokine stimulus is almost totallydisappeared in lymphocytes.7. The nonhuman animal model lacking the function to control lymphocytemigration according to any of paragraphs 1 to 6, wherein marked atrophyof lymphoid follicles, straying of lymphocytes into red pulp, anddisappearance of marginal-zone B cells are observed.8. The nonhuman animal model lacking the function to control lymphocytemigration according to any of paragraphs 1 to 7, wherein the nonhumananimal is a mouse.9. A method for screening a promoter or suppressor of the function tocontrol lymphocyte migration, wherein a test substance is administeredto the nonhuman animal model lacking the function to control lymphocytemigration according to any of paragraphs 1 to 8, or a test substance isbrought into contact with tissues, organs or cells from said nonhumananimal model and a wild-type animal to measure/assess the change in thefunction to control lymphocyte migration.10. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to paragraph 9, wherein thechange in the function to control lymphocyte migration is a change inthe active GTP-bound Rac.11. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to paragraph 9, wherein thechange in the function to control lymphocyte migration is a change inthe migration activity of lymphocytes in response to stimuli withchemokines such as SLC, SDF-1, BLC, etc.12. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to paragraph 9, wherein thechange in the function to control lymphocyte migration is a change inthe homing activity to secondary lymphoid organs such as spleen, lymphnodes, Peyer's patches and the like.13. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to paragraph 9, wherein thechange in the function to control lymphocyte migration is a change inthe number of mature T cells in peripheral blood in response tochemokine stimulus with ELC, or a change in the intra-thymic migrationactivity of CD4⁺CD8⁺ immature thymocytes in response to chemokinestimulus with SDF-1.14. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to paragraph 9, wherein thechange in the function to control lymphocyte migration is a change inthe degree of actin polymerization in lymphocytes in response tochemokine stimuli.15. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to paragraph 9, wherein thechange in the function to control lymphocyte migration is a change inthe degrees of atrophy of lymphoid follicles, straying of lymphocytesinto red pulp, and disappearance of marginal-zone B cells.16. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to any of paragraphs 9 to 15,wherein the test substance is a molecule which binds to DOCK2.17. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to any of paragraphs 9 to 16,wherein the degrees of change in the function to control lymphocytemigration of a nonhuman animal lacking DOCK2 gene on the chromosome andof a wild-type nonhuman animal are compared and assessed.18. The method for screening a promoter or suppressor of the function tocontrol lymphocyte migration according to any of paragraphs 9 to 17,wherein the nonhuman animal is a mouse.19. A promoter or suppressor of the function to control lymphocytemigration obtained by the method for screening according to any ofparagraphs 9 to 18.20. The suppressor of the function to control lymphocyte migrationaccording to paragraph 19, wherein said suppressor is an anti-DOCK2antibody, a DOCK2-binding molecule or an antisense strand of DOCK2 gene.21. A method for screening a therapeutic agent for immune-relateddiseases such as allergy, antoimmune diseases, GvH, graft rejections,wherein the method for screening a promoter or suppressor of thefunction to control lymphocyte migration according to any of paragraphs9 to 18 is used.22. A therapeutic agent for immune-related diseases such as allergy,autoimmune diseases, GvH, graft rejections obtained by the screeningmethod according to paragraph 21.23. The therapeutic agent for immune-related diseases according toparagraph 22, which is an anti-DOCK2 antibody, a DODK2-binding moleculeor an antisense strand of DOCK2 gene.24. A protein for controlling lymphocyte migration which mediatesreorganization of cytoskeleton through activating Rac.25. The protein for controlling lymphocyte migration according toparagraph 24, wherein said protein is DOCK2 and a DOCK2 variant.26. The protein for controlling lymphocyte migration according toparagraph 25, wherein DOCK2 is an expression product of Hch gene(GenBank: accession No. AYO27438).27. A method using the protein according to any of paragraphs 24 to 26for controlling lymphocyte migration.28. DNA encoding a protein for controlling lymphocyte migration whichmediates reorganization of cytoskeleton through activating Rac.29. DNA encoding the protein for controlling lymphocyte migrationaccording to paragraph 28, wherein said DNA is DOCK2 gene and a DOCK2gene variant.30. DNA encoding the protein for controlling lymphocyte migrationaccording to paragraph 29, wherein DOCK2 gene is Hch gene (GenBank:accession No. AYO27438).31. A method using DNA according to any of paragraphs 28 to 30 forexpressing the protein for controlling lymphocyte migration.

INDUSTRIAL APPLICABILITY

The present invention revealed that DOCK2, a haematopoieticcell-specific CDM family protein, is indispensable for lymphocytechemotaxis. DOCK2-deficient mice (DOCK2^(−/−)) exhibited migrationdefects of T and B lymphocytes, but not of monocytes, in response tostimuli with chemokines, resulting in abnormalities including Tlymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zoneB cells. In DOCK2⁻/⁻ lymphocytes, chemokine-induced Rac activation andactin polymerization were almost totally disappeared. Thus, DOCK2 isshown to control lymphocyte migration by functioning as a main moleculethat mediates cytoskeletal reorganization through Rac activation. Forthis reason, by using the animal model of the present invention,immune-related diseases such as allergy, autoimmune diseases, GvH andgraft rejection, or the pathogenic conditions can be elucidated at amolecular level and a novel therapy for these diseases or pathogenicconditions can be developed by targeting DOCK2.

1. A method for screening a suppressor of lymphocyte migration activity,comprising the following steps (a) to (c): (a) activating Rac with DOCK2by stimulating lymphocytes with one or more chemokines in vitro in thepresence or absence of a test substance; (b) performing at least one ofthe following steps (b1) to (b3): (b1) detecting a GTP-bound activatedRac; (b2) measuring a change in actin polymerization; and (b3) measuringlymphocyte migration; and (c) determining the test substance to be aDOCK2-mediated suppressor of lymphocyte migration activity when at leastone of Rac activation, actin polymerization, and lymphocyte migration inthe above step (b) is suppressed in the presence of the test substanceas compared to a case in the absence of the test substance.
 2. A methodfor screening a suppressor of lymphocyte migration activity, comprisingthe following steps (a) to (d): (a) searching for a test substance thatbinds to DOCK2; (b) activating Rac with DOCK2 by stimulating lymphocyteswith one or more chemokines in vitro in the presence or absence of thetest substance searched in the above step (a); (c) performing at leastone of the following steps (c1) to (c3): (c1) detecting a GTP-boundactivated Rac; (c2) measuring a change in actin polymerization; and (c3)measuring lymphocyte migration; and (d) determining the test substanceto be a DOCK2-mediated suppressor of lymphocyte migration activity whenat least one of Rac activation, actin polymerization, and lymphocytemigration in the above step (c) is suppressed in the presence of thetest substance as compared to a case in the absence of the testsubstance.